Chapter title |
3D d STORM Imaging of Fixed Brain Tissue.
|
---|---|
Chapter number | 13 |
Book title |
Synapse Development
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6688-2_13 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6686-8, 978-1-4939-6688-2
|
Authors |
Frank Herrmannsdörfer, Benjamin Flottmann, Siddarth Nanguneri, Varun Venkataramani, Heinz Horstmann, Thomas Kuner, Mike Heilemann |
Editors |
Alexandros Poulopoulos |
Abstract |
Central nervous system tissue contains a high density of synapses each composed of an intricate molecular machinery mediating precise transmission of information. Deciphering the molecular nanostructure of pre- and postsynaptic specializations within such a complex tissue architecture poses a particular challenge for light microscopy. Here, we describe two approaches suitable to examine the molecular nanostructure of synapses at 20-30 nm lateral and 50-70 nm axial resolution within an area of 500 μm × 500 μm and a depth of 0.6 μm to several micrometers. We employ single-molecule localization microscopy (SMLM) on immunolabeled fixed brain tissue slices. tomoSTORM utilizes array tomography to achieve SMLM in 40 nm thick resin-embedded sections. dSTORM of cryo-sectioned slices uses optical sectioning in 0.1-4 μm thick hydrated sections. Both approaches deliver 3D nanolocalization of two or more labeled proteins within a defined tissue volume. We review sample preparation, data acquisition, analysis, and interpretation. |
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